The structural determinants of the quaternary structure of the multifunctional anthranilate synthase-phosphoribosyl transferase complex of tryptophan biosynthesis in Salmonella typhimurium are being studied. This enzyme aggregate is composed of two molecules each of two dissimilar subunits (components I and II) which are coded by the first two genes (trpE and trpD) of the trp operon. The unaggregated subunits can be readily purified from appropriate mutants and are capable of spontaneous assembly in vitro forming the intact tetrameric complex. A genetic approach to the identification and mapping of the structural regions of the trpE polypeptide involved in its assembly with the trpD polypeptide is being applied. This involves the isolation of an extensive set of trpE missense mutants in which the affinity of component I for component II is either reduced or totally eliminated. A refinement of the current fine structure map of trpE is being made by quantitative recombinational analyses of a large number of auxotrophs. An estimation of the number and location of assembly sites in the component I molecule will then be made by mapping the isolated assembly-defective mutations. Purification and characterization of the catalytic, conformational and assembly properties of representative mutant component I subunits will then follow. Characterization of the kinetics of assembly of the wild type complex from homogenous component I and II subunits is underway to complement the mutant study. The effects of the following parameters are being analyzed: subunit concentration; temperature; the composition, pH, ionic strength and viscosity of the medium; and the presence of specific ligands of the two subunits. Assembly is being monitored by changes in the fluorescence properties of the subunits as well as by the appearance of glutamine-dependent AS activity, a property characteristic of the complex.